Introduction

Breast cancer is the second leading cause of cancer death in women

worldwide, and is expected to surpass heart diseases in the next few years (Torre et
al., 2016; Siegel et al., 2015). It accounts for around 7% of global burden of cancer
and 1/5th of all the cancers in India (Boyle and Howell, 2010). According to American
Cancer Society, an estimate of 29% incidences and 15% deaths due to breast cancer
around the world (Siegel et al., 2014) has been predicted. In 2015, 2,31,840 new cases
of invasive breast cancer were diagnosed, resulting in 40,290 deaths in the United
States (Siegel et al., 2015). In India, breast cancer was the leading cancer among
females (24.85%) with the highest incidence and death rates being 10.53 and 16.18
%, respectively (Jivarajani et al., 2015). It has overtaken cervical cancer to become
the leading cancer in metro cities in India and is expected to double in 2016
(Swaminathan et al., 2011). It has been estimated that by 2030, the global incidence of
breast cancer would be increased to more than 2 million new cases per year, however,
in India it would reach up to 2 lakh per year (Ferlay et al., 2010).

Breast cancer is a clinically heterogeneous disease with multi-factorial

etiology, caused due to several risk factors including environmental, hormonal,
dietary, lifestyle, and genetic factors; exposure to the ionizing radiation; as well as
race, age, gender, ethnicity, and family history (Martin and Weber, 2000). Breast
cancer management is a major clinical challenge due to its complexity, heterogenecity
and aggressiveness (Kimbung et al., 2015). The conventional treatments available for
breast cancer include surgery, chemotherapy, radiation therapy, hormonal therapy and
targeted therapies (Perez et al., 2011; Howard et al., 2012). Even though these
treatments are highly effective, yet they are associated with serious side effects
(Binkley et al., 2012) that have shifted the global focus towards Complementary and
Alternative Medicines (CAM). Use of CAM has become increasingly popular among
the breast cancer patients throughout the world (Wanchai et al., 2010). It was
estimated that the use of CAM in cancer patients varied from 7- 64% with higher use
(47-83%) in breast cancer patients (Link et al., 2013).

Herbal medicine that forms an integral part of CAM has been reported to play
a key role in the management of breast cancer (Liao et al., 2013). Different medicinal
plants including Podophyllum peltatum (Mayapple), Taxus baccata (Pacific Yew),
Chapter-1 Introduction

Vinca rosea (Periwinkle) and Camptotheca acuminate (happy tree) have been
evaluated in clinical trials for breast cancer (Mantle et al., 2000). Medicinal plants are a
source of a large number of bioactives that are excellent anticancer agents as they have the
efficiency to regulate the molecular mechanisms and various signalling pathways involved in
carcinogenesis such as oxidation, inflammation, apoptosis, cell proliferation, cell cycle,
invasion, metastasis and angiogenesis (Albulescu, 2015).

Even though medicinal plants and their bioactives have been reported to be
highly potent anticancer agents, the widespread use of herbal bioactives has been
restricted due to their hydrophobic nature that reduces their bioavailability and in turn
reduces their therapeutic efficacy (Ahmad et al., 2006; Opara and Chohan, 2014).
This problem has been overcome with the advent of nanotechnology, which has made
a significant impact on the development of novel drug delivery systems (NDDS)
(Bonifácio et al., 2014; Gunasekaran et al., 2014). Much efforts has been made to use
modern nanotechnology to deliver herbal drugs (Bhadoriya et al., 2011) for safer and
more effective breast cancer treatment.

One of the emerging strategies has been the use of plant extracts for
synthesizing metal nanoparticles (such as gold and silver) for anticancer applications
(Makarov et al., 2014; Kulkarni et al., 2014). Biosynthesis of gold nanoparticles has
been reported by using extracts of hibiscus (Philip, 2010), oat and wheat (Armendariz
et al., 2002), geranium (Shankar et al., 2003), lemon grass (Shankar et al., 2005),
tamarind (Ankamwar et al., 2005), bengal gram (Ghule et al., 2006), cinnamon
(Huang et al., 2007), tea (Nune et al., 2009), neem (Shankar et al., 2004), cumin
(Katti et al., 2009), Aloe vera (Chandran et al., 2006), onion (Parida et al., 2011) and
many more (Ahmed and Ikram, 2015).

Other strategies that have been used since long time involve conjugation of
anticancer bioactives with nanocarriers such as liposomes, micelles, polymeric
nanoparticles, metal nanoparticles, dendrimers and solid lipid nanoparticles (SLNs) to
obtain enhanced therapeutic efficacy (Padmavathi, 2013; Bonifácio et al., 2014).
Various herbal bioactives such as berberine (Ma et al., 2013), vincristine (Abe et al.,
2011), thymoquinone (Odeh et al., 2012), topotecan and quercitine (Zucker and
Barenholz et al., 2010), shikonin (Kontogiannopoulos et al., 2012), artemisinin
(Dadgar et al., 2013), silibinin (Ebrahimnezhad et al., 2013), oridonin (Wang et al.,
2014), paclitaxel (Dilnawaz et al., 2010), camptothecin (Min et al., 2008), docetaxel
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Chapter-1 Introduction

(Mei et al., 2009), tryptanthrin (Fang et al., 2011) and many others have been
conjugated with these nanocarriers for drug delivery applications in breast cancer. The
nanocarriers have significantly improved the bioavailability of herbal bioactives
compared to their free counterparts at in vitro, in vivo and preclinical level (Watkins
et al., 2015). A number of leading pharmaceutical companies, specialized in the
production of high quality personalised botanical extracts and bioactives drugs have
introduced standardized herbal nanoformulations (Verma et al., 2014). Various
patents have been filed for herbal drug tagged nanoparticles having good stability,
enhanced biodistribution and efficacy and reduced toxicity (Jadhav et al., 2014).

Based on the above context, the broad aim and objective of this thesis is to
explore the potential of nanoparticles in the delivery of herbals or their bioactives in
breast cancer cells. The overall work in the thesis involves synthesis, characterization
and biological studies of nanoparticles conjugated with herbals as drugs against breast
cancer. In this study, two metal nanoparticles (gold and iron oxide nanoparticles) were
synthesized.

Iron oxide nanoparticles were chemically synthesized by co-precipitation of

ferric and ferrous salts, followed by tagging with bioactive molecules present in Butea
monosperma and Camellia siunensis with citric acid was used as a linker that is
known to inhibit glycolysis, which is used by cancer cells to survive. It was
hypothesized that its use could further enhance the anticancer activity of curcumin
synergistically. Glycine was used as a linker in cinnamaldehyde conjugated iron oxide
nanoparticles as its amine group could be used to functionalize the iron oxide
nanoparticles. Pluronic F127 was also used for functionalization of NPs since it has
been known to be a versatile polymer for water dispersibility and would generate
additional hydrophilicity and stability to the system.

The synthesized nanoparticles were characterized for their shape, size, phase
purity and bonding by various microscopic and spectroscopic techniques. Drug
loading and release profiles, stability and biocompatibility of the nanoparticles were
also studied. All the three nanoparticles were found to be in biological size range of 1-
100 nm. The nanoparticles were further explored for their activity against breast
cancer cell lines, MCF-7 and MDA-MB-231, and two non-cancerous cell lines, HEK
293 and MCF10A. Both breast cancer and non-cancerous cells were not affected by

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Chapter-1 Introduction

the treatment of F-AuNPs indicating that the phytoconstituents of Ficus religiosa

provide a non-toxic coating onto the gold nanoparticles. Since NPs were not toxic to
breast cancer cell lines, they were not taken up for further studies. The lack of any
noticeable toxicity provides new opportunities for their possible applications in
molecular imaging. Decreased the survival of breast cancer cells however with similar
IC50 values as that of pure curcumin. NPs decreased the viability of breast cancer cell
lines at lower doses of cinnamaldehyde in comparision to pure cinnamaldehyde,
thereby implying improved bioavailability and in turn therapeutic efficacy. NPs were
non-toxic to non-cancerous cells and were found to reduce the growth of breast cancer
cells in a dose and time dependent manner. NPs were further studied for their effect
on the cellular molecular mechanisms underlying their anticancer activity as these
NPs had higher drug payload and more cytotoxicity towards breast cancer cells,
compared to NPs. NPs altered migration, reduced expression of angiogenesis marker
and induced apoptosis in breast cancer cells. Further, upon exposure to the
radiofrequency waves, NPs heated up to 41.6°C within 1 min, suggesting their
promise in magnetic hyperthermia application.

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